Concepts Flashcards
Southern Blot
+: Only way to identify some genomic rearrangements (F8)
-: Laborious, Expensive, Large amounts of DNA required
PCR
+: Versatile, can detect mutations directly or facilitate other methods
-: Is an amplification procedure; >1000kb is difficult
ASO Hybridization
+: Can detect single base-pair substitutions; can be parallelized, automated
-: Time Consuming
Allele-Specific PCR
+: Rapid, Can detect single-base substitutions
Multiplex-PCR
+: Rapidly identify large deleted regions
-: Not quantitative, will miss other mutations
MLPA
+: Rapidly Identify large deleted regions, Quantitative
-: Laborious design of probe pairs
FISH (BAC Probe)
+: Can detect large deleted regions and other chromosomal abnormalities
-: Time-consuming; poor resolution
CGH
+: Can identify large imbalances in genomic content
-: Can’t detect balanced rearrangements; poor resolution
DNA Sequencing (Sanger)
+: Can detect virtually any mutation, known or unknown
-: Expensive and very laborious on larger sequences
Allele Tracking
+: Allows genotyping of individuals without knowledge of mutation
-: requires a linked marker, and informative pedigree, and known phase
Wild Type
Polymorphism
Variant
Rare Variant
Wild Type: most common
Polymorphism: > 1% frequency in population
Variant: Change; may/may not be polymorphic or pathogenic
Rare Variant: <1% frequency in population
Mutagenesis
Process by which stable change in the genetic information of an organism occurs.
- Spontaneous Mutations occur for 1st time and are not inherited
- Mutagens increase rate of mutagenesis
- DNA repair increases mutation rate 100 fold
Mutation Types:
- Substitutions
- Deletions
- Insertions
Substitutions: Silent, Missense, Nonsense (null)
Deletions: Multiple of 3, Frameshift, Partial Deletion, Whole gene deletion
Insertion: Same as Deletions + Dynamic Mutation
Forward/Backward Slippage
Backward slippage: Synthesized strand increases (insertion)
Forward Slippage: Synthesized Strand decreases (deletion)
Backwards - Synthesized strand slips
Forwards - Template strand slips
Mode of Inheritance and Mutations
Recessive: Loss of Funciton
Dominant: Haploinsufficiency (null alleles), Dominant Negative (different missense mutations do not yield distinct phenotypes), Gain of Function (do yield distinct phenotypes)
LCRs of Hemoglobin
Embryonic DNA: Epsilon
Fetal: G-Gamma
Adult: Delta or Beta
Haploinsufficiency
Expression in one copy of a gene is insufficient. Loss of function in one gene will show dominant phenotype (complete penetrance)
Ex. TF, Porphyrias, Marfan Syndrome, Type 1 OI
Gain of Function Mutation
Increased or novel gene function (mutant protein synthesized) from Missense (usually), gene duplications, chromosomal translocations.
Different mutations in same gene results in different phenotypes.
Ex. TD1/2, Achondroplasia, Hypochondroplasia and the FGFR3 gene
Dominant Negative Mutation
Mutant gene product interferes with function of normal gene (usually from a missense mutation). Usually associated with structural proteins like collagen.
Ex. OI Type 2 or 3 (Better to have null than missense)
PTC
Premature Termination Codon
- causes RNA product to be degraded (results in Null mutation, NOT truncated protein product)
Duplication and Deletion
Phenotypes are just about the opposite of each other.
Ex. Williams Syndrome vs. Speech Delay (7q11.23), CMT1A vs. HNPP
Static vs. Dynamic Mutations
Static: Stably transmitted to offspring and retained in somatic tissues throughout development
Dynamic: Continue to mutate during transmission to offspring and during tissue development (may cause mosaicism)
Trinucleotide Repeats and Disorders
3bp tandem repeats found within genes (exons, introns, 5’ UTR, 3’ UTR)
CGG 5’ UTR - Fragile X Syndrome (>200 repeats)
GAA Intron - Friedrich Ataxia (200-1700 repeats)
CAG Exon - Huntington’s (36-121 repeats), SCA (39-82 repeats), others
CTG 3’ UTR - Myotonic Dystrophy (50-4000 repeats)
Anticipation
Slippage in one person causes increasingly severe symptoms as genes are passed down (also earlier onset each generation)
Most are due to Expansions (backwards slippage)
Germline-Specific Repeat Instability
Paternal Expansion Bias: CAG expansions in Huntington’s, SCA
Maternal Expansion Bias: CGG in Fragile X, CTG in Myotonic Dystrophy
Trinucleotide Repeat Disorders and Mutation types.
LOF (Null)
- Friedrich Ataxia (Auto Recessive)
- Fragile X (X-linked Dom)
Gain of Function (Altered RNA function)
- Myotonic Dystrophy I and II
Gain of Function (Altered Protein Function)
- Huntington’s
- SCA
SRY (Sex-Determining Region Y)
Primary determinant of male of sexual development
46 XX with SRY+ (Gain of Function)
- Infertile Phenotypic Males (due to missing Y genes for spermatogenesis)
46 XY with SRY- (Loss of Function)
- Infertile Phenotypic Females (due to needing 2 X chromosomes for oocyte maintenance)
X Chromosome Inactivation
Done to compensate for dosage difference of X chromosome between males and females. Is always random and results in mosaicism in females.
Mechanisms of Epigenetics
DNA Methylation
- Cytosines in CG dinucleotides; methylation = silencing
Histone Modification
- Acetylation - activating
- Methylation - Variable
ATP-Dependent Chromatin Remodeling
- Swi/Snf - Variable
Histone Variants
- Canonical H2A, H2A.Z, H2A.X - variable
Noncoding RNA
- miRNA, siRNA, IncelRNA
CpG Islands
DNA sequences with high CG frequency. Often upstream of genes. Hypermethylation = strong silencing
Mutation hotspots (most frequent single nucleotide mutations occur here): G -> A (mismatch repair), C -> T (deamination of methylated C)
Genetic Imprinting
Inactivation of specific autosomal genes when inherited from gender-specific parents.
Note: the imprinting pattern is inherited in somatic DNA, but when an offspring produces their own gametes, the imprint ‘resets’ and will adjust to the gender of the offspring producing the gametes.
Ex. Prader-Willi (SNRPN), Angelman’s (UBE3A) (15q11-13)
Uniparental Disomy; Isodisomy and Heterdisomy
NDJ and subsequent Trisomy Rescue mechanism can cause a imprinting disorder because both imprinted genes can be inherited from one parent (1/3 chance) if the Trisomy Rescue removes the gene from the other parent to fix the NDJ.
Isodisomy: NDJ in Meiosis II
Heterodisomy: NDJ in Meiosis I
Hydatidiform Mole vs. Ovarian Teratoma
Hydatidiform Mole: 2 Paternal Genes
- vigorously growing membranes, no embryo
- extraembryonic
Ovarian Teratoma: 2 Maternal Genes
- disorganized fetal body parts, no membranes
- embryonic
Gene Identification
Functional Cloning: ID a gene by its product
- Ex. Globins, Factor 8, Enzymes
Positional Cloning: ID a gene by its location (uses linkage of markers to disease alleles)
- Ex. Huntington’s, CF, Achondroplasia
Linkage
Tendency for loci to be transmitted together as an intact unit through meiosis.
Linkage analysis: method of mapping genes by measuring how close a gene is to a known marker. Distance is estimated by determining how often recombination occurs between marker and gene.
Recombination Frequency
= Number of Recombinant Progeny/Total number of progeny
Genetic Distance measured in centiMorgans (cM)
RF = 0.01 = 1% = 1 cM
Population Genetics
Study of distribution and change of frequency of alleles within populations.
Genetic, Societal, Environmental factors that change/maintain frequencies.
Diseases associated with deficiency of HEXA enzyme.
Tay-Sachs
- Mutations in HEXA (encodes alpha subunit of HEXA)
Sandhoff
- Mutations in HEXB (encodes beta subunit of HEXA)
Activator Deficiency (AB Variant) - Mutations in GM2A (encodes activator protein, very rare)
IDENTICAL CLINICAL PRESENTATIONS
Assumptions for Hardy Weinberg Law
- Large Population
- Random Mating
- Constant Gene Frequency
- No effect of recurrent mutation
- no selection against a phenotype
- no migration in or out of the population
Disruptions of Hardy Weinberg Eq
Exceptions to Random Mating
- Stratified Mating (I like Korean girls more than other girls)
- Assortative Mating (Positive and Negative) (I like light-skin because I have light skin, I like short girls because I am tall)
- Consanguineous Mating (Incest)
Exceptions to Large Population
- Genetic Drift (Dying in small population without offspring reduces frequency of your allele combination)
- Founder Effect (Founders of new colony bring carrier traits from source population and those traits are retained as population grows. Ex: Ashkenazi Jews and Tay-Sachs)
Exceptions to Constant Allele Frequency
- Gene Flow (mutation spreads radially from site of origin)
- Heterozygote advantage (Sickle Cell Trait gives immunity to Malaria)
- Selection against mutant alleles due to loss-of-fitness
Penetrance
High: Mendelian (single gene)
- Other genes and environmental factors have no effect
- Ex. Huntington’s
Low: Multifactorial
- each single gene plays a small part along with other genes/environmental factors
- Ex. Multiple Sclerosis
Polygenic/Oligogenic
Polygenic: many genes each having limited impact on their own on the phenotype. Some have large effects (called susceptibility genes)
Oligogenic: Few Genes
Measurement of Traits
Quantitative: continuous varability within a limiting range (height, body mass)
Qualitative (Dichotomous): present or absent (you have a disease or you don’t)
Liability
Susceptibility.
Liability threshold model: You cross a threshold, you have whatever it is you’re liable to have. The higher above the threshold, the greater the severity of the disease.
Sibling Risk Ratio
= Prevalence of disease in siblings of affected individual/Prevalence of diseases in the general population
If a disease has no genetic component, gamma = 1.
if gamma > 1, there is a genetic component.
Concordance
Two related individuals share a trait.
Rates: proportion of pairs of individuals that share a trait (easiest to use with qualitative traits)
100% MZ Twin concordance and 50% DZ Twin = likely a genetic contribution
If MZ < 100%, there is evidence for nongenetic factors
Genetic Liability in family members is increased in a specific scenario.
- affected individual has a severe form of the disease
- affected individual is of a gender that normally does not contract disease.
- more than one affected individual in the family
Screening for Genetic Disease
High Risk (positive Family History) vs. Low Risk (General Population)
Three types:
- Prenatal Screening/Diagnosis
- Noninvasive = Screening
- Invasive = Diagnostic - Newborn Screening
- Carrier Screening
Prenatal Screening (Noninvasive)
FIRST (1st trimester aka 11-14 weeks)
Maternal Serum Screening (2nd trimester aka 15-20 weeks)
Ultrasound
FIRST
Check for hCG and PAP-A in maternal serum
Ultrasound for Nuchal Translucency
Maternal Serum Screening
Triple Screen/Test
- AFP
- uE3
- hCG
If you want to improve detection of Down’s Syndrome, add check for:
4. Inhibin-A
Not as accurate as FIRST
Prenatal Diagnosis (Invasive)
Chorionic Villus Sampling (10-14 weeks)
Amniocentesis (16-20 weeks)
Cordocentesis (19-21 weeks LAST RESORT if amniocentesis and ultrasound fail to give conclusive results)
ALL are associated with elevated risk of inducing miscarriage. Perform as early as possible to allow for termination of pregnancy in first trimester
